CONFORMATIONAL AND DYNAMIC CHANGES OF YERSINIA PROTEIN-TYROSINE-PHOSPHATASE INDUCED BY LIGAND-BINDING AND ACTIVE-SITE MUTATION AND REVEALEDBY H D EXCHANGE AND ELECTROSPRAY-IONIZATION FOURIER-TRANSFORM ION-CYCLOTRON RESONANCE MASS-SPECTROMETRY/

Protein tyrosine phosphatases (PTPase) play important roles in the int racellular signal transduction pathways that regulate cell transformat ion, growth, and proliferation. Here, solvent accessibility is determi ned for backbone amide protons from various segments of wild-type Yers inia PTPase in the presence or absence of 220 mu M vanadate, a competi tive inhibitor, as well as an active site mutant in which the essentia l cysteine 403 has been replaced by serine (C403S). The method consist s of solution-phase H/D exchange, followed by pepsin digestion, high-p erformance liquid chromatography, and electrospray ionization high-fie ld (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry . Proteolytic segments spanning similar to 93.5% of the primary sequen ce are analyzed. Binding of vanadate reduces the H/D exchange rate thr oughout the protein, both for the WpD loop and for numerous other resi dues that are shielded when that loop is pulled down over the active s ite on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the struc ture in several other segments. Although the 3D structure of the ligan d-bound C403S mutant is similar to that of the wild-type PTPase, and t he C403S mutant and the wild-type enzyme display similar affinities fo r vanadate, the thermodynamics for binding of vanadate is different fo r the two proteins. Collectively, these results establish the flexibil ity of the WpD loop (previously inferred by comparing PTPase X-ray sin gle-crystal diffraction structures in the presence and absence of a tu ngstate inhibitor), as well as several other significant changes in se gment exposure and/or flexibility that are not evident from X-ray stru ctures.